Biology subjects

Shevchik, V. E.

Publications and source records attributed to Shevchik, V. E..

4 recordsLinked to original sources

bioRxiv2026

Architecture of the type II secretion system

The virulence of emerging Gram-negative pathogens frequently arises from toxins delivered by the type II secretion system1. Cryo-EM single particle analysis and cryo-electron tomography and have defined the outer membrane secretin pore in detail, but the organisation of proteins within the periplasm and inner membrane that form the pilus assembly platform is not well resolved2,3. Here we combine AlphaFold4 models with single particle cryo-EM to define the organisation of the pilus assembly platform. We show that CLM heterotrimers form a continuous link from the cytoplasmic ATPase, across the inner membrane and periplasm, to the base of the secretin channel. AlphaFold models of the inner membrane spanning rotor and cytoplasmic ATPase fit readily within the cryo-EM density. The resolved secretion system exhibits an offset between the inner membrane assembly platform and the outer membrane secretin pore, together with profound asymmetry and an unexpectedly open periplasmic architecture. This architecture provides a route by which large, folded proteins access the secretion channel from the periplasm and suggests that substrate engagement may trigger the final steps in secretion system assembly leading to secretion.

microbiology↗

bioRxiv2026

Structural determinants of endopilus assembly, stability and functional specificity in bacterial type II secretion

Gram-negative bacteria employ the type II secretion system (T2SS) to transport folded protein effectors across the outer membrane. This multiprotein nanomachine assembles the endopilus, a periplasmic helical polymer composed of one major and four minor pilin subunits. Endopili are structurally related to type IV pili but exhibit distinct features including a conserved calcium-binding site stabilizing their major pilin subunits. Endopilus polymerisation is coupled to substrate translocation through a dedicated outer membrane channel. To investigate the structural basis of endopilus assembly, stability and functional specificity, we performed a comparative analysis of the Out T2SS from the plant pathogen Dickeya dadantii and the Pul T2SS from the human pathogen Klebsiella oxytoca. Although their major pilins OutG and PulG share over 77% sequence identity, these bacteria differ markedly in ecological niche and range of secreted effectors. We report here the NMR structure of calcium-bound OutG monomer and cryo-EM structures of OutG and PulG endopili at 3.6 [A] resolution. The integration of structural, mutational, and biophysical analyses with in vivo assays, allowed us to identify the molecular determinants of secretion specificity and endopilus stability. These findings demonstrate how subtle sequence variations in conserved nanomachines have evolved to optimize their function and adapt to their local environments.

microbiology↗

bioRxiv2024

Inner membrane protein OutB is covalently attached to peptidoglycan in the γ-proteobacterium Dickeya dadantii

Gram-negative bacteria possess a multilayered envelope comprising an inner membrane (IM), a thin peptidoglycan (PG) layer, and an outer membrane (OM). In Escherichia coli and certain other {gamma}-proteobacteria, Braun lipoprotein (Lpp) covalently tethers the OM to PG. Only a few other OM proteins have been found to be covalently linked to PG in Gram-negative bacteria. Here, we showed that in the phytopathogenic {gamma}-proteobacterium Dickeya dadantii, an IM protein, OutB, is covalently attached to PG, thereby tethering itself and the associated type 2 secretion system to the cell wall. In contrast to Lpp, OutB reaches the PG layer from the IM side. By modifying the length of Lpp, which would displace the PG layer in the periplasm, we found that the elongated Lpp+21 improved OutB attachment to PG, whereas the shortened Lpp{Delta}21 reduced it. We showed that two L,D-transpeptidases, Ldt03 and Ldt84, tether Lpp and OutB to PG by the same catalytic mechanism involving the formation of an amide bond between their C-terminal lysine and the stem peptide. Ldt03 and Ldt84, each display substrate specificity for the type of peptide stem and preferentially cross-link Lpp to monomeric and dimeric muropeptide, respectively. The C-terminal Lpp-like box of OutB is almost identical to that of Lpp; it tolerates substantial amino acid substitutions and allows PG-tethering of a bona fide periplasmic protein. Thus, it seems possible that the repertoire of periplasmic and membrane proteins tethered to PG may be more extensive than currently assumed.

microbiology↗

bioRxiv2021

Scaffolding protein GspB/OutB facilitates assembly of the Dickeya dadantii type 2 secretion system by anchoring the outer membrane secretin pore to the inner membrane and to the peptidoglycan cell wall.

The phytopathogenic proteobacterium Dickeya dadantii secretes an array of plant cell wall degrading enzymes and other virulence factors via the type 2 secretion system (T2SS). T2SSs are widespread among important plant, animal and human bacterial pathogens. This multi-protein complex spans the double membrane cell envelope and secretes fully folded proteins through a large outer membrane pore formed by 15 subunits of the secretin GspD. Secretins are also found in the type 3 secretion system and the type 4 pili. Usually, specialized lipoproteins termed as pilotins assist the targeting and assembly of secretins into the outer membrane. Here, we show that in Dickeya, the pilotin acts in concert with the scaffolding protein GspB. Deletion of gspB profoundly impacts secretin assembly, pectinase secretion, and virulence. Structural studies reveal that GspB possesses a conserved periplasmic Homology Region domain that interacts directly with the N-terminal secretin domain. Site-specific photo cross-linking unravels molecular details of the GspB-GspD complex in vivo. We show that GspB facilitates outer membrane targeting and assembly of the secretin pores and anchors them to the inner membrane while the C-terminal extension of GspB scaffolds the secretin channel in the peptidoglycan cell wall. Phylogenetic analysis shows that in other bacteria, GspB homologs vary in length and domain composition and act in concert with either a cognate ATPase GspA or a pilotin GspS. ImportanceGram-negative bacteria have two cell membranes sandwiching a peptidoglycan net that form together a robust protective cell envelope. To translocate effector proteins across this multilayer envelope, bacteria have evolved several specialized secretion systems. In the type 2 secretion system and some other bacterial machineries, secretins form large multimeric pores that allow transport of effector proteins or filaments across the outer membrane. The secretins are essential for nutrient acquisition and pathogenicity and constitute a target for development of new antibacterials. Targeting of secretin subunits into the outer membrane is often facilitated by a special class of lipoproteins called pilotins. Here, we show that in Dickeya and some other bacteria, the scaffolding protein GspB acts in concert with pilotin, facilitating the assembly of the secretin pore and its anchoring to both the inner membrane and the bacterial cell wall. GspB homologs of varied domain composition are present in many other T2SSs.

microbiology↗